Further characterization of the ppGalNAcT family: To date, 16 human ppGalNAcT isoforms have been described in the literature. Jaya Raman has conducted an extensive search of human and mouse nucleotide sequence databases that revealed the existence of an additional 4 human isoforms which she is characterizing (together with other members of the lab) with respect to expression pattern, and relatedness to known family members by sequence analysis and enzymatic activity. In a manuscript that is being revised, we will assign unique names to each of these novel isoforms and propose names for several previously described mammalian ppGalNAcTs to eliminate confusion present within the nomenclature of current sequence databases and the ppGalNAcT literature. Molecular mechanisms of ppGalNAcT catalysis: Some members of the ppGalNAcT family demonstrate preference for substrates that are already decorated with GalNAc (i.e. glycopeptides). In collaboration with Tom Gerkin Tim Fritz and Jaya Raman emplyed a series of random peptide and glycopeptide substrates to evaluate the substrate specficities of ppGalNAcT-10 (which prefers glycopeptides) and ppGalNAcT-1 and T-2 (which prefer peptides). ppGalNAc T10 exhibited strong preference for Ser/Thr-O-GalNAc at the +1, C-terminal position relative to a Ser/Thr acceptor site and no preferences for specific amino acids. Biological consequences of ablating expression/activity of ppGalNAcTs: In collaboration with Stasia Anderson, Daryl Despres and M Starost from NIH, Yu Guan has studied the cardiac phenotypes resulting from the deletion of ppGalNAcT-1. She found that the heart/body weight ratio was increased in 4-month-old KO animals compared to heterozygotes and wild-type littermate controls. Histological analysis revealed right or left ventricle hypertrophy, enlarged cardiomyocytes, increased interstitial fibrosis, and mitochondria swelling and dissolution in knockout animals. Further analysis revealed that the heart valves are markedly enlarged in the affected hearts. Yu is analyzing the underlying molecular cause(s) for this phenotype. Hazuki Miwa has demonstrated by real-time PCR, that ppGalNAcT-1 (T1) transcripts are highly expressed in bone suggesting the involvement of T1 in bone protein glycosylation. The apparent molecular masses of bone proteins extracted from wild-type and T1-knockout mice were determined by SDS-PAGE and western blot analysis to reveal that osteopontin (OPN) and bone sialoprotein (BSP) from T1-knockout migrated further than did those from the wild-type. After mucin-type O-glycans were enzymatically removed, the apparent molecular masses of OPN and BSP from the wild-type and T1KO were reduced to the same size, suggesting that the observed molecular mass differences were due to incomplete glycosylation in T1-knockout mice. While T1-knockout mice lack an obvious bone phenotype, OPN and BSP play a variety of functional roles. Thus, the effects of T1-mediated glycosylation on their functions will be investigated. We continue to collaborate with Suzanne Walker (Harvard) to identify small molecule inhibitors of ppGalNAcTs. Yu Guan is performing the biological assays on cell lines with candidate inhibitors. We continue to collaborate with Dr. Danielle Dube, Bowdoin College on the characterization of a novel yeast-two hybrid system that she developed which challenges protein interactions within the Golgi rather than the nucleus.